I made use of the known dates of reclamation (and of afforestations) in the IJsselmeerpolders in The Netherlands to assess evolutionary adaptation in Cepaea nemoralis. At 12 localities (three in each polder), I sampled a total of 4390 adult individuals in paired open and shaded habitats, on average 233 m apart, and scored these for genetic shell colour polymorphisms. The results show (highly) significant differentiation at most localities, although the genes involved differed per locality. Overall, though, populations in shaded habitats had evolved towards darker shells than those in adjacent open habitats, whereas a 'Cain & Sheppard' diagram (proportion yellow shells plotted against 'effectively unbanded' shells) failed to reveal a clear pattern. This might suggest that thermal selection is more important than visual selection in generating this pattern. Trait differentiation, regardless of whether they were plotted against polder age or habitat age, showed a linear increase of differentiation with time, corresponding to a mean rate of trait evolution of 15-31 kilodarwin. In conclusion, C. nemoralis is capable of rapid and considerable evolutionary differentiation over 1-25 snail generations, though equilibrium may be reached only at longer time scales.Heredity advance online publication, 14 November 2012; doi:10.1038/hdy.2012.74.

One of the best known and most intensively studied model systems for selection on colour
polymorphism is the land snail Cepaea. In Cepaea nemoralis and C.
hortensis, two common European species, very conspicuous, and heritable (Murray, 1975), variation exists in shell colour and colour
pattern. Shell ground colour ranges from bright yellow to deep brown and superimposed on
this a variable pattern of dark brown spiral bands may exist (Lang,
1904; Goodhart, 1987). Numerous studies have
shown that natural selection acts strongly on many of these colour morphs under different
circumstances (though C. nemoralis often differs from C. hortensis in
the exact response; Clarke, 1960). Evidence exists for
selection for habitat-dependent cryptic colouration by predation by the song thrush,
Turdus philomelos (Cain and Sheppard, 1950,
1954; Lamotte, 1951;
Carter, 1968). On the other hand, the effects of shell
colour on the snails' thermoregulation (Heath, 1975)
create a separate selection pressure, leading to summertime selection for shells with
higher albedo in exposed habitats (Cain, 1968; Jones et al., 1977) and lower latitudes (Jones et al., 1977; Silvertown
et al., 2011), and wintertime selection for light-coloured shells
in topographies that are prone to extreme cold events (Cameron,
1969). In addition, some effects of shell colour on behaviour have also been
reported: Wolda (1965) and Jones
(1982) found that different colour morphs have different tendencies to climb
trees, and to rest in exposed sites. Finally, phylogeographic patterns in allele
predominance (so-called ‘area effects') exist that may not be related to any
direct environmental selection (Cain and Currey, 1963;
Davison and Clarke, 2000). Because of this mixture of
evolutionary forces and the spatial scales at which they operate, it has often been
difficult to untangle the precise mechanism for observed colour differentiation (Jones et al., 1977; Cook,
1998).

This habitat-related selection in Cepaea is often quoted as an example of
contemporary, though relatively slow (requiring at least 100 generations; Cook, 1998) evolution. However, only in a few studies are the ages
of the populations and/or the habitats known with some certainty, allowing
evolutionary rates to be estimated. For example, Richards and Murray
(1975) made use of a C. nemoralis population introduced into North
America, and demonstrated that, in this population fixed for yellow shell ground colour,
shaded habitat populations had obtained significantly higher banding and higher band
fusion frequencies over a period of at least 43 years. Similarly, Ożgo and Bogucki (2011) performed a transplant experiment in Poland,
showing that, over 11 years, frequencies of yellow and yellow effectively unbanded in
C. nemoralis diverged slightly but significantly between open and shaded
habitats. Ożgo and Kinnison (2008) detected strong
shell colour divergence in three open/sheltered habitat pairs in southeastern Poland
that had presumably been established 18–28 years previously. Ożgo (2011), finally, surveyed 12 newly-established open/shaded
habitat pairs in Germany and Poland (presumably 60–80 years old) and concluded that
most of these showed significantly higher frequencies of yellow and lower frequencies of
band fusion in the open habitats.

These studies suggest that, with a generation time of around 3 years (Jones et al., 1977; Cook,
1998), Cepaea populations can diverge under habitat-associated
selection in time spans much less than 100 generations. However, it may be argued that
larger series of time-controlled (natural) experiments with known dates of population
establishment are needed for a proper assessment of evolutionary rates in Cepaea.
Here, I adopt the Dutch IJsselmeerpolders to provide such a system. These large areas of
reclaimed land (polders) around the former Zuiderzee estuary were drained in the
mid-20th century and subsequently colonised by flora and fauna, including C.
nemoralis. As the C. nemoralis populations inhabiting paired
open/shaded habitats in these polders cannot be older than the polders themselves, and
as the four major polders were drained with 11–15 intervening years, they allow
evolutionary rates to be estimated with some accuracy.

Materials and methods

Genetics and scoring system for the Cepaea shell colour
polymorphism

Murray (1975) summarises the genetics of the shell
colour polymorphism in C. nemoralis. The locus C controls shell ground
colour, with alleles that determine colours ranging from pale yellow to deep brown (in
increasing dominance). The shell may also carry one to five dark brown spiral bands,
which are sometimes fused with one another. Banding is controlled by the B
locus (linked with C). The dominant allele (for unbanded) suppresses a
recessive allele for banding. At the unlinked T locus the dominant allele
suppresses bands 1 and 2, leaving only the three lowest bands. The dominant allele at
the U locus removes bands 1, 2, 4 and 5, making shells mid-banded. All banding
categories with the top two bands absent are termed ‘effectively unbanded'.
The polygenic genetic control of band fusion, finally, is not yet resolved. Shell
phenotypes are conventionally coded with a letter (Y, P or B) for the ground colour
(yellow, pink and brown), followed by five digits (1–5), each replaced by a zero
for a missing band. Band fusions (determined a quarter of a whorl away from the
aperture) is coded with parentheses around the band digits that are joined. As an
example, the following morphs are all effectively unbanded pink: P00000, P00300, P00345,
P00(345), P003(45) and P00(34)5.

Study areas and sampling

The IJsselmeerpolders comprise four large areas in the north-central part of The
Netherlands (see Figure 1): Wieringermeer (WI;
193 km2, drained in 1930), Noordoostpolder (NOP;
469 km2, 1942), Oostelijk Flevoland (OF; 528 km2,
1957) and Zuidelijk Flevoland (ZF; 430 km2, 1968). Today, the areas
consist mostly of agricultural land, but also large industrial and residential areas
exist, with scattered wetland and woodlands. To select habitat pairs, I used satellite
images available in Google Earth and visited many suitable habitat pairs in the winter
of 2010/2011, and, based on the availability of sufficient empty Cepaea
shells, selected 12 pairs (three in each polder; see Figure
1 and Supplementary Table 1). Distances
between the two habitats in a pair averaged 233 m (range: 35 to 650 m) and
were thus less than the minimum of 1 km recommended by Cameron and Cook (2012a). Using the historical aerial photographs available
at http://historische-luchtfoto.flevoland.nl/, editions of topographic maps
spanning much of the 20th century, and interviews with local foresters, I also estimated
the year (in most cases±3 years) in which each shaded habitat had appeared. In
the remainder of this paper, I shall use the term ‘locality' for each of the
12 places where habitat pairs were sampled, the term ‘site' for a single
habitat plot, and ‘sample' for the snails collected in a site.

The sites were sampled between 11 May and 23 September, 2011. At each site,
characteristics of the vegetation were recorded, and adult C. nemoralis snails
were collected in a systematic fashion within a plot of <5000 m2. I
aimed to obtain at least 150 individuals from each of the 24 sites. Colour morphs were
scored in the field, after which the snails were released back into the site. No attempt
was made to quantify bird predation. Data were submitted to the online Cepaea
polymorphism database Evolution Megalab (www.evolutionmegalab.org). Bird data show that all sites fell
within areas with high densities of the song thrush (van Diermen,
2002). The soil at all sites consists of young marine clay with a high
density of mollusk shells, except for Voorsterbos, which is on a fluvioglacial deposit
(Wolters-Noordhoff, 2001).

Analyses

For each sample, I calculated the proportions (for all snails) of yellow, pink and
brown, and unbanded, as well as effectively unbanded. I also calculated the proportions
of all band combinations (of which 00300, 00345 and 12345 were the most abundant) among
the banded category, and a measure for band fusion by taking the proportion of 5-banded
snails with at least two bands fused. Differences in morph frequencies within each
habitat pair were tested with χ2-tests. The euclidean distances
generated from the morph frequency data were used to generate a phenogram for all
samples using Ward-method in PAST 2.13 (Hammer et al.,
2001). The pattern in frequencies yellow and effectively unbanded was
inspected via a ‘Cain and Sheppard diagram' (Cook,
2008).

In addition, for each snail, a ‘darkness score' was determined. For this, I
applied the data in Heath (1975) on the thermal
properties of different colour morphs as follows. Using yellow unbanded as the base
line, the expected temperature increases of other morphs were calculated by adding
0.3 °C for pink, 0.6 °C for brown, 0.07 °C for each
band and 0.03 °C for each band fusion. The resultant expected temperature
increase was used as ‘darkness score'. Differences in darkness between two
sites of the same locality were tested for significance with Kruskall–Wallis
one-way analysis of variance, and the overall significance was tested with
Fisher's combined probability test.

Finally, for each habitat pair, I calculated a standardised measure for differentiation
between the open and the shaded habitat for each of the major morph categories (that is,
frequencies of P, B, YeU, banded, eU, 00300, 00345, 12345, fusion in 5-banded and fusion
in all banded). I did this by taking the absolute value of
log(PO/PS), in which PO is the frequency of the
morph concerned in the open habitat, and PS in the shaded habitat. These
differentiation measures were then correlated with polder age (see Figure 1) and estimated age of the shaded habitat (Table
1). Significance was assessed with an F-test. Under the assumption that the
open habitat represented the original morph frequency and the shaded habitat represented
the differentiated state, evolutionary rate was calculated (and expressed in
kilodarwins; Haldane, 1949) as (ln PS−ln
PO)/t for all major morph categories (where t is the
time period in 103 year).

Results

In most sites, I obtained samples of around 150 individuals or more, except for Nagele
Open and Shaded and Cirkelbos Open, where numbers were just under 100. Total number of
snails collected was 4390. The cluster analysis (Figure 2)
shows some phylogeographic patterns: for five localities (Knarbos, Wilgenreservaat,
Nagele, Zuiveringsveld and Cirkelbos) do the open and shaded samples of a pair cluster
together. Also, there is a slight indication of area effects at the scale of polders;
except for Dijkgatsbosch Open, the WI samples all fall within one branch of the phenogram.
Similarly, the frequency of B appears to be low in ZF compared with the other polders
(Table 1).

The morph frequencies (Table 1) show that most habitat pairs
differ significantly for three or more morphs, except for Cirkelbos and Zuiveringsveld.
However, most morphs do not show a consistent habitat pattern. In shaded habitats, there
seem to be tendencies for Y and 12345 to increase, and for P and 00300 to decrease. Other
morphs show about as many increases as decreases, or show no differences. The only
consistent response is in fusion: all seven significant changes in band fusion are towards
more fusion in the shaded habitat. The lack of a consistent response in other shell morphs
is also apparent in the Cain and Sheppard diagram (Figure 3).
When shell colour is converted to a general darkness score, however, patterns become
clearer (Figure 4). In nine cases (seven of which are
significant), shells in shaded habitats have a higher darkness score, and in only three
(two significant) the pattern is reversed. The overall pattern of a change toward darker
shells in shaded habitats is significant (P<0.001; Fisher's combined
probability test; χ2=115; d.f.=24). Finally, open vs
shaded habitat morph frequency difference shows positive and significant correlation with
habitat and area age (Figure 5), leading to mean evolutionary
rates of 14.7 (s.d. 20.7) and 31.4 (s.d. 45.2) kilodarwin (using polder age and habitat
age, respectively).

Discussion

Upon reclamation, the soil that developed in the IJsselmeerpolders provided a suitable
habitat for terrestrial mollusks because of calcium-rich marine clay (Jansen, 2010). It may thus be expected that open habitats suitable for
Cepaea began to appear naturally very soon after reclamation was completed.
Shaded habitats also appeared quickly, as forest was planted from saplings grown for this
purpose at nurseries either within the polders themselves or on the ‘old land'
(Reinink, 1979). However, it appears that Cepaea
was not introduced to the polders during this afforestation process, and in fact, only
appeared many years after reclamation. In 1959, the species could not be found in
grassland, woodland, nor the tree nurseries in OF, 2 years after reclamation (Jansen, 2010). Blaauw (1974), who
surveyed land snails in ZF 5 years after reclamation, also records Cepaea as
absent. Finally, Reinink (1979) failed to find
Cepaea in ZF and OF in 1973/1974 (5–6 and 16–17 years after
reclamation, respectively). He searched all tree nurseries and recently planted plots
thoroughly and, finding no snails at all, concluded that the phytosanitary measures were
very strict.

It thus appears that Cepaea has colonised the polders slowly and under its own
power. It is therefore likely that it has entered planted woodlands after advancing
through open habitat for several generations. More importantly, this means that any
differences between populations in paired open and shaded habitats probably indeed evolved
as these habitats appeared and that shaded habitats were not stocked with populations
pre-adapted to woodland habitats. The pattern of colour morph variation in the polders
suggests that these colonising populations retained sufficient genetic diversity to adapt
to newly-arising environments, and also that some geographical structuring is present. In
some cases, the populations from open habitat are, despite divergent evolution (see
below), more similar in morph frequencies to the shaded habitat in the same locality than
to other populations inhabiting open habitats.

The results show, for most localities, strong differentiation in several morph
frequencies between open and shaded habitat (Table 1). When
converted to a general darkness score, a significant tendency in the expected direction
exists, namely towards darker shells in shaded habitats (seven significant cases), with a
minority (two significant cases) showing the reversed response. Three cases (Nagele,
Robbenoordbos and Zuiveringsveld) show no significant difference in darkness.

Absence of differentiation may be due to a rate of gene flow that is too great for
differentiation to build up. In the case of Nagele, this is a plausible explanation, as
the open habitat is a narrow strip of reedland immediately adjacent to the woodland (at a
distance of the same magnitude as the per-generation dispersal in C. nemoralis;
Jones et al., 1977). At Robbenoordbos and
Zuiveringsveld, however, this cannot be the case, as the two habitats, which have existed
for several decades, are separated by 650 and 180 m, respectively, and, in both
cases, by a road and two ditches. It is therefore not clear what may have prevented
differentiation at these two localities.

The pattern for the remaining localities, of a majority of cases with darker shells in
shaded habitat and a minority of cases with a reversed pattern, has been encountered
before (Ożgo, 2005; Cook,
2008; Cameron and Cook, 2012a). It is generally
assumed that the combined effects of visual selection and thermal selection tend to work
in the same direction, that is, towards darker shells in the shaded habitat, but may
sometimes work in opposite directions, depending on the alleles present in the population,
the exact vegetation structure (which differed within the open and shaded habitat
categories), and the season in which most selection takes place (Jones et al., 1977). As a consequence of the relative strengths of
thermal and visual selection pressures, this may sometimes lead to lighter shells in the
shaded habitat.

Nonetheless, the majority of the IJsselmeerpolder localities show darker shells in the
shaded habitat. The shell morphs responsible, however, differ strongly per locality. For
example, at Kathedralenbos, yellow is decreased dramatically in favour of pink. At
Kolhorn, yellow is also decreased, but here in favour of brown. At Voorsterbos, yellow is
actually increased, but this is more than compensated by a greater number of banded
snails. The only morph change that seems to be consistent across all localities is towards
more band fusion in the shaded habitat. A similarly predictable response of band fusion
was also found by, for example, Ożgo (2011). In fact,
surprisingly, the categories most often reported to show consistent responses to habitat,
that is, yellow and effectively unbanded, show no indication of a consistent response
(Cain and Sheppard diagram; Figure 3). Cameron and Cook (2012b) similarly found that the habitat separation in a
Cain and Sheppard diagram is only clear in Southern England and disappears on large parts
of the European mainland. As changes in these colour morphs are most often thought to be
associated with visual selection, it may be argued that the evolutionary changes that we
do see in the present study are caused primarily by thermal selection, rather than visual
selection. In this respect, it must also be pointed out that the inheritance and
heritability of band fusion (the trait that contributes most consistently to shell
darkness in this study) is far from clear (Lang, 1904;
Murray, 1975), and further experiments to elucidate this
are needed.

The chief aim of this paper was to examine the rate of evolutionary change, by making use
of the known ages of the polders and the habitats in them. Interestingly, significant
positive correlations exist between morph frequency differentiation and age, both for the
age of the entire polder and for the respective habitats. Ages are given in years, but as
generation time in C. nemoralis is c. 3 years (Jones et al., 1977; Cook, 1998), the
range equates with a time scale of 1–25 snail generations. Over this time scale,
evolutionary change in shell colour traits appears to accumulate with a mean rate of
15–31 kDar, and Figure 5b suggests that this
process has not yet stabilised. It should be emphasised that these are minimum rates: the
(limited) data on colonisation history suggest that the polders were not colonised
immediately, and, hence, snail populations may, in some cases, have been established
considerably later than the polders and/or the habitats, or there may have been a
history of extinction and recolonisation. It should also be emphasised that the
evolutionary change is not just the result of habitat-related selection, but probably
include other kinds of genetic differentiation (for example, founder events and genetic
drift) as well. Future work on comparisons of genetic differentiation across the same
habitats in different polders and vice versa might enable separation of these multiple
effects.

The rates of evolutionary change found here may be considered high (Kinnison and Hendry, 2001; Bell, 2008), and
they clearly show that, indeed, as reported by Ożgo
(2011), Ożgo and Kinnison (2008) and
Ożgo and Bogucki (2011), Cepaea is capable
of very rapid adaptive divergence in shell colouration in response to different habitat
conditions. At the same time, the fact that evolutionary divergence seems to be greatest
in those habitat pairs that have existed around 80 years (27 generations), it is not
impossible that divergence will reach equilibrium only on a time scale close to 100
generations, as suggested by Cook (1998).

I thank Małgorzata Ożgo for sharing her vast knowledge of Cepaea
biology with me and for providing the pre-defined spreadsheets that form the basis for
Supplementary Table 1. Jan Akkerman and Ruben Kluit
(Natuurmonumenten), Ed Colijn (European Invertebrate Survey—The Netherlands), Roelof
Duijff (Flevolandschap), and Harco Bergman (Staatsbosbeheer) provided crucial information on
the history of the polders and their habitats. Three anonymous referees provided comments
that considerably improved the paper.

Ożgo M,Bogucki Z. Year: 2011Colonization, stability, and adaptation in a transplant experiment of the
polymorphic land snail Cepaea nemoralis (Gastropoda: Pulmonata) at the edge of
its geographic rangeBiol J Linn Soc104462470